1. Field of the Invention
The present invention relates to an improved method for quantitative measurement of liquid samples using a dry type procedure (multilayer analysis film technology), more particularly it is concerned with a method for assaying liquid samples especially effective for the quantitative determination of viscous liquid samples such as whole blood that spread poorly when the samples are spotted on the multilayer film.
2. Development of the Invention
Multilayer analysis films are already known which can be used to determine chemical components (especially components that increase the viscosity of the system, e.g., glucose, total protein, triglycerides, etc.) contained in a liquid sample with ease and with high speed via a dry type procedure. For instance, specific examples of such analysis sheets are described in Japanese Patent Application (OPI) No. 53888/74 (corresponding to U.S. Pat. No. 3,992,158), No. 137192/75 (corresponding to U.S. Pat. No. 3,983,005), No. 40191/76 (corresponding to U.S. Pat. No. 4,042,335), No. 3488/77 (corresponding to U.S. Pat. No. 4,006,403), No. 131786/77 (corresponding to U.S. Pat. No. 4,056,898), No. 131089/78 (corresponding to U.S. Pat. No. 4,144,306), No. 29700/79 (corresponding to U.S. Pat. No. 4,166,093) and No. 34298/79 (corresponding to British Pat. No. 2,000,869A) (the term "OPI" as used herein refers to a published unexamined Japanese patent application), U.S. Pat. Nos. 4,110,079 and 4,132,528, Clinical Chemistry, vol. 24, pages 1335 to 1350 (1978), and so on.
Such multilayer analysis elements have a common format wherein a spreading layer capable of spreading a liquid sample, layers containing reagents essential to the analysis elements and so on are multi-coated in advance on, a support and, upon the actual chemical analyses using these sheets, quantitative analysis can be conducted through two basic processes: spotting of the sample liquid into the sheet and evaluation of the extent of dye formation by a specific reaction using a densitometer. These processes are referred to as dry chemical analyses as they do not require processes which are indispensable for conventional methods, such as: arrangement of test tubes; preparation, volume measurement and addition of reagent solutions; accurate weighing-out of samples; and so on.
The basic structure of the multilayer chemical analysis sheet of the type described above comprises a support, a reagent layer and a sample spreading layer, which are arranged in this order.
A liquid drop of a liquid sample dropped onto a spreading layer of the analysis sheet is uniformly diffused over the spreading layer and, at the same time, permeates into a reagent layer, where an analyte contained in the liquid drop is converted into a detectable chemical species (normally color formation or a color change occurs). By observation and quantitative determination of the degree of color formed or the color change, an analyte contained in the liquid sample can be quantitatively assayed.
A major reason for such dry type analysis procedures being operable with good precision is because a liquid sample-spreading layer is arranged to supply a liquid sample to a reagent layer at an approximately constant volume per unit area. Depending upon the volume of the liquid sample applied, this layer acts as a spreader for the liquid sample. In order to obtain a spreading layer having excellent capability for spreading a liquid sample, a variety of materials have recently been studied and developed. In the case of using a liquid sample having high viscosity, especially whole blood or preserved blood having a markedly high hematocrit value as a liquid sample, however, a satisfactory spreading action can be obtained only with difficulty with conventional materials for spreading layers, and regions in which measurement was substantially impossible were present. Taking into account the fact that detection of a disease factor of an abnormally high concentration (also highly viscous) is principally practical in the field of clinical examination, it has been desired that substantially undetectable regions should be minimized as much as possible and the detection range should be broadened as much as possible.
In the prior art, a method in which a highly viscous liquid sample is previously diluted to a suitable region prior to analytical operations (hereafter simply often referred to as "simple dilution") has been adopted. According to this simple dilution method, the degree of dilution can be controlled depending upon the viscosity of a liquid sample so that it must be theoretically possible to measure an analyte utilizing a spreading action of a multilayer analysis film, even though a liquid sample having a high vicosity is employed. However, the level (concentration of color formed or changed) of a detectable signal which is to be formed in a reagent layer is reduced in inverse proportion to the degree of dilution as the degree of dilution of a liquid sample becomes high, and, as a result, there is the danger that optical reading might be inaccurate.
Furthermore, it is difficult to accurately weigh small quantities of a blood sample, particularly having a high hematocrit value and a high viscosity; accordingly, error due to dilution tends to be serious. An error in volume in collecting a sample is particularly serious when small quantities of less than 100 .mu.l are weighed using a micropipet, etc. Particularly when an amount of a liquid sample is less than several ten times (twenty to fourty) .mu.l, it is no exaggeration to say that quantitative weighing would be impossible unless a particular device is used and the severest possible attention is paid. Further, in actual dilution, a considerable amount of water must be added to a liquid sample (for example, dilution of a 5 .mu.l liquid sample using 5 .mu.l of water is substantially impossible).
In addition, it is required that dilution be carried out outside the reaction system and, as a result, additional processes are added to the measurement procedure. Furthermore, test tubes are required for dilution procedures. Moreover, quantitative determination of an analyte is impossible unless dilution is so accurate that one can determine the degree of dilution. This is because color formed or changed which is actually detected is of the thus diluted liquid sample and the absolute amount cannot be determined unless the degree of dilution is considered. These procedures thus reduce the most advantageous features involved in multilayer analysis films which do not require precise measurement of a definite volume of a liquid sample and permit simple measurement. It has thus been desired to develop a quantitative assay method without impairing these advantages of multilayer analysis films.
One method for improving spreading comprises treating the surface of a liquid sample-spreading layer with a surface active agent, thereby assisting spreading of a liquid sample when the liquid sample is dropped on the spreading layer; such is described in Japanese Patent Application (OPI) No. 131786/77 (U.S. Pat. No. 4,050,898) and No. 164356/80. Further, a method which comprises physically activating a material for a spreading layer by a glow discharge treatment, etc., to thereby assist spreading of a liquid sample is described in Japanese Patent Application No. 140532/80 (OPI No. 66359/82).
In these methods, however, a liquid sample is applied to a multilayer analysis film which is almost completely dried and, as a natural consequence, spreading is not rapidly facilitated since the multilayer analysis film is firstly wetted and then spreading follows. It is known that when surface active agents or highly hygroscopic components such as glycerin or the like are incorporated in a spreading layer, the spreading property is improved; however, the high water content of multilayer analysis materials generally results in the disadvantages of poor reagent stability during storage, undesired color formation (fogging during storage), etc. Thus, in practice there is a limit for imparting hydrophilicity and even in the case that multilayer analysis films having provided the thus improved spreading layer are employed, highly viscous liquid samples that involve disadvantages such as poor spreading, a prolonged period of time for spreading, etc., still exist, and it has been desired to develop multilayer analysis films which can cope with any liquid sample.